The purpose of this grant is to investigate basic immunological components and anatomical/biochemical properties of the eye with regard to the distribution and regulation of humoral elements, such as immunoglobulins (Ig) and complement, and to investigate their role(s) in immune-complex (IC) mediated ocular inflammation elicited by foreign, exogenous (non-self) antigens. The normal Ig class/subclass distribution will be determined within different human and mouse intraocular tissues using immunoperoxidase techniques. The charge and quantity of Ig class/subclasses will be determined by ELISA, Immunoblotting, and Isoelectric Focusing of ocular tissue eluates. These studies will provide information regarding the potential regulation of intraocular Ig distribution by electrostatic gradients. The intraocular distribution of specific antibody will be analyzed by specialized staining techniques of murine tissues to determine the experimental kinetics of the appearance and disappearance of antibodies of different class/subclass and charge. These data will be correlated with serum titers and will help to delineate the sources and bioavailability of specific antibodies of varied biological properties and activities. The Ig and antibody distribution studies will be correlated with similar studies investigating the localization of complement components within the uvea and sclera. The accessibility of circulating antigens to these and other intraocular tissues will be investigated using both endogenous (plasma) and exogenous tracer antigens (horseradish peroxidase or hapten-carriers). Histochemical, biochemical, and immunochemical, studies will be undertaken to analyze intraocular glycosamminoglycan (anionic) gradients which may regulate the intraocular interactions of these charged molecules. A murine model of uveal in situ IC formation and induced inflammation will be utilized to investigate different parmeters which regulate the biological properties of IC formed within the eye. The charge and class/subclass, as well as the affinity of antibodies within the IC will be investigated both polyclonal and monoclonal antibodies. Analogous studies will examine the role(s) of antigen charge, molecular weight, and valence in intraocular IC formation. Subsequent experiments will examine the complement requirement in IC mediated ocular inflammation using C5 deficient mice and the C3 inhibitor cobra venom factor. Additional experiments will investigate mechanisms of IC clearance and resulting changes within the eye.